Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method

Fluorescent labeling allows for imaging and tracking of vesicles down to single-particle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility. Here, we describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, we show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. We consistently observed 6- to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.

were collected from the interface between the sucrose cushion. For bacterial OMVs, cultured 23 lysogeny broth from E. coli W3110 cells grown until the OD600 reached 1.5 was centrifuged twice 24 at 6,000×g to remove cells. The supernatant was filtered through 0.45-μm pores and was 25 concentrated with a QuixStandTM benchtop system using 100-kDa hollow fiber membranes 26 (Amersham Biosciences). The concentrated supernatant was filtered through 0.22-μm pores, and 27 then OMVs were pelleted by ultracentrifugation at 150,000×g for 2 h at 4 °C. The EVs and OMVs 28 were further purified through iodixanol buoyant density gradient ultracentrifugation. 29

Preparation of PD-1-GFP-loaded NK-92 EVs 30
To construct an NK-92 cell line expressing PD-1-GFP, we purchased a lentivirus vector with 31 cDNAs for murine PDCD1 gene (NM008798) and mGFP (Origene MR227347L4): 32 Then, a lentivirus was constructed by applying the lentivirus vector encoding PD-1-GFP. After 59 treating the virus on NK-92 cells, polybrene (Millipore) was added to a final concentration of 8 60 μg/ml and spinoculation was performed at 360×g for 90 min at 32 °C to increase infection 61 efficiency. Infected NK-92 cells were sorted for the overexpression of GFP using MoFlo Astrios 62 EQ (Beckman Coulter) and cultured in NK-92 complete culture medium maintained with 1 μg/ml 63 puromycin. EVs were collected in the same way as described for NK-EVs without PD-1. 64

Nanoparticle tracking analysis (NTA) 71
For NTA measurements, vesicle samples were diluted with filtered PBS to ~10 9 particles/ml and 72 illuminated with 405-nm laser in the NTA equipment (Nanosight, LM10-HS). About 30 73 measurements were performed to analyze each vesicle sample. 74 Labeling with PKH67 using a standard protocol 75 For labeling with PKH67 following a standard protocol ("Protocol Guide: Exosome Labeling 76 Using PKH Lipophilic Membrane Dyes" on sigmaaldrich.com), NK-CDVs were first pelleted by 77 centrifugation at 18,000×g for 80 min. The pellets were resuspended with 100 µl of Diluent C 78 (Sigma, CGLDIL), mixed with 0.6 μl of PKH67 dye (Sigma, MIDI67) by gentle pipetting, and 79 5 incubated for 5 min at room temperature. The reaction was quenched by adding 200 µl of 10% 80 bovine serum albumin (BSA) (Biosesang, A1025) in PBS, and the total volume of the sample was 81 increased up to 1 ml by adding Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, D6429). 82 The labeled CDVs were collected and excess dyes were cleared by centrifugation at 18,000×g for 83 2 h at 4 °C. The pellets were resuspended in 900 µl of PBS and 75 µl of DMEM. The sample was 84 purified and concentrated again using an Amicon filter (10-kDa cutoff, Millipore, UFC5010) at 85 3,000×g for 40 min. After purification, the labeled CDVs were diluted to 100 µl with PBS. A 86 sample with CDV-free media was prepared by the same procedure for the negative control. 87

TIRF microscopy and image analysis 88
Sample slides for TIRF imaging were prepared as previously described. 4 Briefly, flow cells were 89 assembled from a glass coverslip and a glass slide bonded together using double-sided tape. Glass were imaged with a custom TIRF microscope (Olympus IX73) equipped with a 60× oil-immersion 99 lens (Olympus) for both illumination and observation. Samples were illuminated by the 488-, 532-100 and 633-nm CW lasers (Cobolt) and the resulting fluorescent images were acquired by an sCMOS 101 6 camera (Teledyne Photometrics, Prime BSI Express) typically with 100-ms resolution and ~100-102 μm field of view. For quantification of fluorescence, initial 10 frames of movies were averaged 103 and either the number of fluorescent spots or the total intensity over the entire area was measured 104 using custom MATLAB codes. 105

Estimation of fluorescent labeling efficiency 106
For photobleaching experiments, the labeled vesicles captured by anti-CD63 were continuously 107 illuminated and the resulting changes in fluorescent intensities from individual spots were tracked. 108 The signal from individual DiI molecules were measured both from the step size during 109 photobleaching events and from the distribution of intensities that showed equally spaced Gaussian 110 distributions. The results from two approaches agreed well and yielded ~65 as the single-dye 111 fluorescence. Finally, the initial intensity from all vesicles were collected (mean ~ 122) and the 112 labeling efficiency was estimated as 122/65 = 1.9. Additionally, the intensity distribution 113 discretized by the step size was well fitted with a Poisson distribution with a mean of 1.9 ( Figure  114 S2E). A single phospholipid molecule is expected to occupy 0.629 nm 2 in lipid vesicles, 5 and the 115 surface area of a small, 100-nm vesicle is 4π×((100 nm)/2) 2 ~ 3×10 4 nm 2 . Therefore, we estimate 116 that each DiI-labeled vesicle contains ~10 5 molecules of lipid in the two leaflets. As a result, the 117 mole fraction of DiI in vesicles (less than 10 molecules per vesicle) in DiI-CDV would be lower 118 than 10 −4 .